: The long-term goal of this research is to elucidate the mechanisms of toxicant/hypoxia-induced cell injury and death. Our laboratory and others have shown that calpains, a family of Ca2 dependent proteases, play a critical role in toxicant/hypoxia-induced cell death. For example, a group of dissimilar calpain inhibitors prevented renal cell death produced by a diverse group of toxicants and promoted the return of respiration and active ion transport following hypoxia/reoxygenation. However, studies to date have not elucidated the mechanism of calpain activation under physiological or pathological conditions, nor have the critical intracellular targets of calpains during cell injury been identified. Previous reports indicate that the level of calpain phosphorylation may be an important determinant of activity. This proposal will test the central hypothesis that calpain dephosphorylation during hypoxic cell injury results in calpain-mediated mitochondrial dysfunction. Specific Aim 1 will determine the level of calpain phosphorylation under control and hypoxia/reoxygenation conditions. In addition the activity of calpains with different levels of phosphorylation will be determined. Specific Aim 2 will discover how calpains mediate mitochondrial dysfunction during hypoxia in situ and in isolated mitochondria, and identify the mitochondrial proteins targeted by calpains using proteomics/mass spectroscopy. Completion of these studies will increase our understanding of events that lead to hypoxic cell death, and aid in the development of therapeutic agents for the treatment of acute renal failure.